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Vol. 20, Issue 4, 1213-1222, February 15, 2009

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GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative and DNA Polymerases in Fission Yeast

Chen Chun Pai^*, Ignacio García^,, Shao Win Wang^*,, Sue Cotterill^||, Stuart A. MacNeill^,¶, and Stephen E. Kearsey^*

*Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom; Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; ^||Department of Biochemistry and Immunology, St. George's Hospital Medical School, SW17 0RE, United Kingdom; and ^¶Department of Biology, University of Copenhagen, Copenhagen Biocenter, 2200 Copenhagen N, Denmark

Submitted April 25, 2008; Revised November 25, 2008; Accepted December 8, 2008
Monitoring Editor: Karsten Weis

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of β-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase , and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase .

Present addresses: Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County, 350, Taiwan;

Departamento de Biología Funcional, Universidad de Oviedo, 33006 Oviedo, Spain.

Address correspondence to: Stephen E. Kearsey (stephen.kearsey{at}zoo.ox.ac.uk).